Anti-CD20 (2H7) h(CD28-CD3ζ) CAR, pSBCAR1 vector

All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

Details

Target
CD20

Targeting Cell Type
T cell

Targeting Diseases
Acute lymphoblastic leukemia (ALL)

Generation
Second

Vector Name
pSBCAR1

Vector Length
~6kb

Vector Type
Sleeping Beauty (SB) transposon

Receptor Construction
scFv-CD28-CD3ζ

Discription of Signaling Cassetes
CD28
CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide costimulatory signals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins which are expressed on antigen-presenting cells (APC). CD28 modulates the primary TCR/CD3ζ signal in a different fashion than the late costimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators that impact on T-cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced sustained T cell activation, growth, survival. And CD28 results in a brightly expressed, stable receptor as the transmembrane domain. Including CD28 costimulatory domains in CARs led to enhanced anti-malignancy efficacy.
CD3ζ
CD3ζ, also known as T-cell receptor zeta, which together with T-cell receptor and CD3γ, δ, ε chain, forms the TCR-CD3 complex. ζ was expressed independently from the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. CD3-zeta, which contains 3 ITAMs, is the most commonly used endodomain component of CARs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.

Target

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Published Data

Complete CAR data FCM

Fig.1 The specific toxicity of anti-CD20 CAR-T cells.

CAR Construction : 2H7-CD28-CD3ζ

Fig.1 The specific toxicity of anti-CD20 CAR-T cells.

HEK293 cells engineered with CD19, CD20 or both, and non-modified HEK293 cells (each 2.5 x 10^4 cells) were stained with CSFE and co-incubated with T cells with the anti-CD20 CAR or a CAR of irrelevant specificity as control.

Martyniszyn, A., Krahl, A. C., Andre, M. C., Hombach, A. A., & Abken, H. (2017). CD20-CD19 bispecific CAR T cells for the treatment of B-cell malignancies. Human gene therapy, 28(12), 1147-1157.

Complete CAR data FCM

Fig.2 The CAR-T mediated T cell cytotoxicity towards Raji cells.

CAR Construction : 2H7-CD28-CD3ζ

Fig.2 The CAR-T mediated T cell cytotoxicity towards Raji cells.

CSFE-labeled Raji cells (5 x 10^4 ) were co-incubated with CAR T cells (10^5 cells) for 24 h.

Martyniszyn, A., Krahl, A. C., Andre, M. C., Hombach, A. A., & Abken, H. (2017). CD20-CD19 bispecific CAR T cells for the treatment of B-cell malignancies. Human gene therapy, 28(12), 1147-1157.

Complete CAR data FCM

Fig.3 The CAR-T mediated T cell cytotoxicity towards primary leukemia cells.

CAR Construction : 2H7-CD28-CD3ζ

Fig.3 The CAR-T mediated T cell cytotoxicity towards primary leukemia cells.

CSFE-labeled patient's CLL cells (5 x 104 ) were co-incubated with CAR T cells (105 cells) for 24 h.

Martyniszyn, A., Krahl, A. C., Andre, M. C., Hombach, A. A., & Abken, H. (2017). CD20-CD19 bispecific CAR T cells for the treatment of B-cell malignancies. Human gene therapy, 28(12), 1147-1157.

CAR scFv data FACS

Fig.4 FACS can profiles of CD20 mAb binding to Raji cells.

CAR Construction :

Fig.4 FACS can profiles of CD20 mAb binding to Raji cells.

Solid profile represents the binding of the fluorescein isothiocyanate-labeled secondary antibody. Open profiles show binding of the primary antibodies 1F5, 2H7, and B1 as indicated.

Deans, J. P., Robbins, S. M., Polyak, M. J., & Savage, J. A. (1998). Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment. Journal of Biological Chemistry, 273(1), 344-348.

CAR scFv data FCM

Fig.5 The binding ability of anti-CD20 mAbs

CAR Construction :

Fig.5 The binding ability of anti-CD20 mAbs

The binding ability of anti-CD20 mAbs using CHO cells stably expressing the wild-type or mutant human CD20, in which amino acids 170 and 172 were both replaced with serin

Uchiyama, S., Suzuki, Y., Otake, K., Yokoyama, M., Ohta, M., Aikawa, S., ... & Fukui, K. (2010). Development of novel humanized anti‐CD20 antibodies based on affinity constant and epitope. Cancer science, 101(1), 201-209.

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.